An unusual di-heme enzyme MauG catalyzes the 6-electron oxidation of a precursor methylamine dehydrogenase with a monohydroxylated 2Trp57 (preMADH) to form the mature tryptophan tryptophyl quinone (TTQ) cofactor. The reaction proceeds via three, two-electron oxidations involving the insertion of the second oxygen atom into 2Trp57, formation of the crosslink between 2Trp57 and 2Trp108, and oxidation to the quinone. The order of these modifications is unknown. MauG can use 3 moles of either H2O2 or O2 plus reducing equivalents to oxidize preMADH. Addition of stoichiometric H2O2 to MauG results in the formation of a catalytically competent bis-Fe(IV) species with one of the hemes in a Fe(IV)=O state while the other is a Fe(IV) species ligated by His and Tyr ligands. This intermediate demonstrates an unprecedented method for stabilizing a highly oxidizing species equivalent to an Fe(V). A crystal structure of the preMADH- MauG complex shows that the site of oxygen binding and activation is over 30 z from the TTQ site. Addition of excess H2O2 to the crystals results in formation of TTQ, demonstrating that the crystals are catalytically active, that each step occurs processively without dissociation of the complex, and that oxidation occurs via long range inter-protein electron transfer. One goal of this project is to structurally characterize the bis-Fe(IV) catalytic intermediate of MauG. The other objective is to characterize the order and nature of each of the 2-electron oxidation reactions occurring at the TTQ site. These biosynthetic intermediates will be generated in crystallo and structurally characterized. Mass spectrometry will also be used to confirm the results of crystallography experiments and to characterize steps involving proton transfer or radical formation, which has been implicated in the first oxidation step. These experiments promise to provide significant insight into methods of radical and high- valent oxidant stabilization within proteins as well as mechanisms of inter-protein electron transfer. These processes underpin aerobic metabolism and have been implicated in various disease states and aging, making them significant to human health.